Plant surfaces are covered with a complex mixture of lipids, which are thought to be synthesized by the cells of the epidermal tissue, the outermost single layer of cells. In addition to functioning as a water barrier, the surface lipids of plants have been suggested to function, inter alia, in frost resistance, in plant-pathogen interactions, and to provide protection from UV irradiation. Despite these diverse and important physiological functions of the surface lipids of plants, little is known about the biochemical and molecular genetic mechanisms that regulate their biogenesis. In particular, only subsequent to the filing of the parent of this application were any of the genes that encode regulatory or enzymatic functions in the cuticular lipid biosynthetic pathway cloned, despite the earlier identification in the art of genes believed to be involved therein.
The outermost barrier of the aerial portions of plants, the cuticle, consists of a meshwork ("cutin") of cross-esterified polymerized hydroxy-fatty acids, which are embedded in a complex mixture of nonpolar lipids, commonly referred to as the cuticular waxes. Maintenance of this interface between a plant and its environment permits sequestration of the highly regulated internal biochemical processes crucial for the plant's survival and the establishment of a water barrier. See Martin and Juniper, The Cuticle of Plants (Edward Arnold Ltd., Edinburgh, UK, 1970); and Kolattukudy, Ann. Rev. Plant Physiol., 32, 539-567 (1981). Chemical analyses of the cuticle have shown that its composition is genetically controlled and is unique to each species. See Kolattukudy and Walton, Prog. Chem. Fats Other Lipids, 13, 121-175 (1973); Kolattukudy et al., in Chemistry and Biochemistry of Natural Waxes (Kolattukudy, ed., Elsevier Press, New York, N.Y., 1976), pages 289-347; Tulloch, in Chemistry and Biochemistry of Natural Waxes, supra, pages 235-287; and Kolattukudy, in The Biochemistry of Plants: A Comprehensive Treatise, Vol. 4 (Stumpf and Conn, eds., Academic Press, New York, N.Y., 1980), pages 571-645.
The biochemistry of cuticular lipid biosynthesis has been investigated by: (1) monitoring the in vivo incorporation of radioactively labeled precursors into the lipids; (2) observing the effect of inhibitors on lipid composition; and (3) determining the in vitro activity of specific enzymes involved in lipid biosynthesis. These investigations have led to the formulation of a hypothesized general scheme of lipid biosynthesis, which is depicted in FIG. 1. See Kolattukudy and Walton, supra (1973); Kolattukudy et al., supra (1976); Tulloch, supra (1980); and Kolattukudy, supra (1980).
The cuticle contains many lipid compounds, the majority of which are derivatives of fatty acids. For example, cutin is composed of fatty acid derivatives such as diols, hydroxy fatty acids, dihydroxy fatty acids and dicarboxylic fatty acids, which are polymerized by ester linkages. The resultant polymers are embedded in the wax components of the cuticle, which is composed of a mixture of hydrocarbons, including n-alkanes, branched alkanes, cyclic alkanes, alkenes, ketones, ketols, alcohols, aldehydes, diols, acids, esters, and the like.
The major components of the cuticular wax, another category of lipids, are very long-chain fatty acids ("VLCFAs") and their derivatives. VLCFAs are considered to be the products of de novo fatty acid biosynthesis, which occurs in plastids, and subsequent elongation by specific elongases that are understood to be localized on the endoplasmic reticulum membranes. Partial purification and characterization of these elongases have been reported. See, for example, Agrawal et al., Arch. Biochem. Biophys., 230, 580-589 (1984); and Agrawal and Stumpf, Arch. Biochem. Biophys., 240, 154-165 (1985). Fatty acyl aldehydes and alcohols are the products of the sequential reduction of the appropriate fatty acid. Wax ester formation probably occurs via a transferase type reaction utilizing fatty acyl-CoAs or phospholipids as the donor of a fatty acid, which is transferred to a fatty alcohol (Kolattukudy et al., supra (1976)). Indeed, a fatty acyl-CoA-fatty alcohol transacylase has been partially purified from young broccoli leaves, and more recently, a similar enzyme has been purified from jojoba seeds (Lardizabal et al., Plant Physiol. (Suppl.), 102, 93 (1993)). As stated previously, prior to the filing of the parent of this application, none of the enzymes involved in cuticular lipid biosynthesis were cloned, accordingly no readily available source of them has been available. Thus, there has existed no ability to use them to alter the cuticular lipid components of a plant or its seed oils, and no ability to use them for in vitro synthesis of the aforementioned lipids.
Maize is a well-characterized experimental plant system that has been the subject of prior investigations of cuticular lipids. The cuticular wax of wildtype maize seedlings is composed of long-chain alcohols (63%), aldehydes (20%), alkanes (1%) and esters of alcohols and long-chain fatty acids (16%). The alcohols and aldehydes are predominantly 32 carbons in length, i.e., n-dotriacontanol (99% of all the alcohols) and dotriacontaldehyde (96% of the aldehydes). The alkane fraction is mainly 31 carbons in length, i.e., hentriacontane (Bianchi et al., Maydica, 30, 179 (1985)). Developmental differences in the quantity and quality of cuticular wax in maize have been noted in that adult maize leaves, as compared to juvenile leaves, have considerably less cuticular wax, which is of a different composition.
Seventeen loci (the glossy or gl genes) have been identified in standard crossbreeding studies of maize as affecting the quantity and composition of cuticular lipids on seedling leaves (namely, glossy1, glossy2, glossy3, glossy4, glossy5, glossy6, glossy8, glossy9, glossy11, glossy14, glossy15, glossy17, glossy18, glossy19, glossy20, glossy21, glossy22), although, as further explained hereinbelow, one of these genes (i.e., glossy15) apparently encodes a developmental control of the activity of cuticular lipid genes. The glossy15 gene also affects cell shape, cell wall composition, and the presence/absence of adult-type epidermal hairs. Hence, it mediates the entire juvenile to adult transition, i.e., it does not regulate cuticular wax biosynthesis per se. See Moose et al., Plant Cell, 6, 1343-1355 (1994) and Evans et al., Development, 120, 1971-1981 (1994).
Mutant seedlings are usually identified because applied water forms droplets on their leaf surfaces (Bianchi, in Maize Breeding and Genetics (Walden, ed., John Wiley and Sons, New York, N.Y., 1978), page 533); in addition, they present a "glossy" appearance. Four of the 17 glossy loci are members of duplicate gene pairs (gl5 & gl20, and gl21 & gl22), i.e., a seedling must be homozygous mutant for both members of such pairs before it expresses the mutant phenotype. Of the 17 glossy loci, 12 have been relatively precisely mapped genetically and three more have been mapped to a chromosome or chromosome arm.
Comparisons of the composition of the waxes produced by seedlings homozygous for each of the various glossy mutants to the wax of wildtype seedlings have been used to identify putatively the biochemical steps encoded by many of the glossy genes. In view of these data, it has been suggested that cuticular wax biosynthesis occurs via two hypothesized pathways, namely ED-I and ED-II (Bianchi et al., supra). The ED-II pathway is thought to be active throughout the life of the plant, with the end product being mainly esters. The ED-I pathway is thought to be active only during the seedling stage of the plant and the end-products of this pathway are mainly alcohols, aldehydes and alkanes.
However, a comparison between the chain lengths of the alcohol moiety of the esters and the chain lengths of the free alcohols in the wax of a number of different glossy mutants suggests an alternative hypothesis that cuticular wax biosynthesis in maize is the result of four reductive systems that are juxtaposed on elongase system(s) (von Wettstein-Knowles, in The Metabolism, Structure, and Function of Plant Lipids (Stumpf et al., Elsevier/North-Holland, Amsterdam, 1987), pages 489-498). One of these reductive systems operates in young seedlings (R1) and appears to produce free alcohols and free aldehydes. The second reductase system (R2) operates in mature plants and catalyzes the esterification of the alcohol products. It has been suggested that the glossy1, glossy8 and glossy18 genes may affect a fatty acid elongation reaction that feeds the R1 reductive system. The glossy7 gene (which is allelic to glossy6), on the other hand, may affect a third reductive system (R3) that provides C28 and C30 alcohols for ester formation. And lastly, according to this interpretation, the glossy15 gene may affect a fourth reductive system (R4) that provides alcohols of even shorter chain length for ester formation. However, more recent analyses of glossy15 have established that glossy15 may not be involved in cuticular wax biosynthesis per se, but rather have an indirect or incidental role in plant cuticle formation, as in the control of the phase change between juvenile and adult leaves (Moose and Sisco, in Abstracts, 35th Annual Maize 1993; and Evans and Poethig, 1993). Mutations at glossy15 result in an early transition from the juvenile to adult stage, resulting in seedling leaves with adult-type wax. Accordingly, it appears that all of the maize glossy mutants are involved in lipid biosynthesis; all but one of these genes (i.e., glossy15) apparently provide the enzymes and, perhaps, the cofactors that mediate lipid biosynthesis. One of the mutants (i.e., glossy 15) apparently relates to the developmental control of the activity of the indicated genes, as well as that of other genes.
Another experimental plant system for the study of cuticular lipids is Arabidopsis. The chemical composition and the genetics of the cuticular lipids of Arabidopsis is less well-defined than for maize. Preliminary analyses of the lipids of Arabidopsis indicate that the composition of the Arabidopsis cuticular wax is distinct from the wax of maize (Hannoufa et al., Phytochem., 33, 851-855 (1993); and Jenks et al., Plant Physiol., 108, 396-377 (1995)). The predominant cuticular lipids of Arabidopsis are alkanes, ketones and secondary alcohols. In contrast to maize, where fatty acyl aldehydes are reduced to primary alcohols, in Arabidopsis a considerable portion of the aldehydes are decarboxylated to form alkanes, which are then further metabolized to ketones and secondary alcohols. Thus, it appears that the Arabidopsis pathway is divergent from that which occurs in maize. However, based on evolutionary considerations, it is considered likely that many of the enzymes involved in the cuticular lipid synthesis of one plant are related in sequence and function to those of another plant. It is understood that fatty acid synthesis occurs in plastids in all plants. Fatty acids of C.sub.16 -C.sub.18 content are transported from the plastid to the cytosol, where it is believed that evolutionarily conserved elongation enzymes act on the de novo fatty acids to form long-chain fatty acids, very long-chain fatty acids, and other lipid compounds. These enzymes are believed to be the same or ancestrally related between species of plants, i.e., they are likely to have related nucleic acid and amino acid sequences.
As with maize, mutations have defined loci that affect the biosynthesis and/or deposition of the cuticular waxes of Arabidopsis. Many of these 21 loci, termed the eceriferum (CER) loci, present readily identifiable phenotypes (Koornneef et al., J. Hered., 80, 118-122 (1989)); some have pleiotropic effects on fertility. Analysis of one of these mutants, at the cer2 gene, indicates that the mutant allele affects changes in the lipid classes and in the predominant carbon chain length of the acyl moieties.
Being at the boundary between the organism and its environment, it would be desirable to control the lipid content of plant cuticle, the biochemical and genetic regulatory mechanisms of which are not well understood. Doing so would facilitate the generation of new plant varieties having altered abilities to conserve water, prevent loss of extracellular components by leeching, and resisting injury from environmental effects such as wind and frost. Because the cuticle is also instrumental in mediating the interaction between plants and other organisms, such as fungi, insects, and bacteria, control of the lipid content of cuticle would also impact on the generation of resistance to these pests. Furthermore, because the nature of the cuticle greatly affects the deposition and behavior of chemicals, including pesticides, growth regulators, and foliar nutrients sprayed on plants, it would be desirable to control the lipid content of cuticles in order to optimize such content for desirable plant variants. It also would be desirable to affect the lipid content of a plant's seeds or to synthesize various plant lipids by in vitro means.
Accordingly, it is an object of the present invention to provide new materials and methods that will allow one to create plant varieties having novel environmental and disease resistances. It is a further object of the present invention to provide new plants having novel environmental and disease resistance and/or plants producing seeds having high levels of saturated or unsaturated long-chain fatty acids and lipids. It is also an object of the present invention to provide plants able to synthesize such fatty acids and lipids of variable and defined chain lengths as well as to provide methods for generating such plants. It is yet a further object of the present invention to provide the isolated genes and gene products responsible for lipid biosynthesis in plants, for in vivo and in vitro synthesis of plant lipids.
These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.